JPH021552A - Detection for decision of diseased condition of person to be examined, monoclonal antibody and detecting kit - Google Patents

Abstract

PURPOSE:To exactly decide the diseased condition such as Kawasaki disease and bacterial infectious disease by detecting the antitumor death factor antibody- reactive material in the bodily fluid taken from a person to be examined by an immunological measurement method using the antitumor death factor antibody. CONSTITUTION:The bodily fluid of the person to be examined under suspicion of being infected by the Kawasaki disease or bacteria infectious disease is taken and the content of the antitumor death factor antibody-reactive material, more specifically the tumor death factor (TNF) contained therein is measured. The content thereof or the change thereof is detected and the progression of the disease condition is judged. The antitumor death factor antibody is used to measure the antitumor death factor antibody-reaction material by the immunological measurement method. The antiserum or monoclonal antibody for the tumor death factor (TNF) is used as the antibody. An ordinarily known sandwich method is used as the method.

Description

DETAILED DESCRIPTION OF THE INVENTION Industrial Application IIIF The present invention provides a detection method for determining the pathological condition of a subject by detecting a substance reactive with anti-tumor necrosis factor antibodies in body fluids of the subject;
The present invention relates to a monoclonal antibody that can be used in the detection method and a detection kit for determining pathological conditions.

To explain in more detail, a detection method for detecting tumor necrosis factor ('I' NF) in a subject's body fluid using an antibody against it and determining the progression of the disease condition, and using the antibody as the antibody. The present invention relates to an anti-'f' NF monoclonal antibody that can be used to detect NF monoclonal antibodies, and a detection kit used in the detection method.

The following tumor necrosis factor ('I'NF) is CD-l5w1ss.
Mouse, Bacillus lus, al+natta-
It was discovered as a physiologically active substance that appeared in the blood when Bacillus Guerin (BCG) was injected, and then bacterial endotoxin was administered two weeks later, and Carswell et al. reported it in 1975 [EA, Carswell et al.
ll et al., Proc. Natl. Acad, S.
ci, USA, 72°3666 (1975)], and its amino acid sequence was published in 1985/
Iggarwal et al. [8, B, Aggarwal et al.
et al. J. Biol, Chen, 260, 2345 (198
5)].

Also Penn1ca at al, 5hirai
et at and WangQtal, human T
The amino acid sequence and gene sequence of NF were revealed [Penn1ca at al Nature 31
2.724 (1985), 5hira+ at al.
Nature 313803 (1985), War
+o et atSiancc 228yo''+49(
1985)]. Initially developed as a cancer treatment drug due to its anti-inflammatory activity, 'l' N-do has recently been revealed to have various physiological activities, and its various functions in vivo are being elucidated6. For example, the activity as an in vivo mesoietor of endotoxin shock caused by bacterial infection [8° Bautler
et al. 5science, 229.869 (1985)],
Induction of inflammatory response in vascular endothelial cells [J, It, Ga
mb l a et al., 1tvoc.

Natl, ^cad, sci,, LISA, 82.8
667 (1985)], exothermic action [C, 8, former nar
ello et al. J, [xp, Hed, bile 3.1443.
(1986), interleukin t 1 pp, which is one of the causative substances of inflammation, NaWrOth et al. J, E
xp, Hed,, 163.1363°f19gG)
] and pseudotaglandins [P, 11. Backwi
ch et al., Biochal, Biophys, l1es
, Con1.136.94 (1986)].

As the results of many such studies become clearer,
``NF is thought to have hormone-like effects as an in-vivo information transmitter that promotes various physiological actions.As a result, the present inventors have investigated the abnormal source, decrease, etc. of TNF in vivo. It has been suggested that changes in TNI? may be related to the pathology of many diseases, and measuring the presence and amount of TNI? in body fluids such as blood is useful for monitoring many diseases. I came to the conclusion that.

On the other hand, according to the research of the present inventors, T N r' is contained in the body fluids of patients with various diseases, and the content of 'f゛N F' and the progress of the patient's disease state are It is believed that there is a deep relationship between It has been found that by taking preventive or therapeutic measures before the disease changes or worsens, it is possible to stop the disease from occurring and sometimes reverse the disease in patients.

In particular, the research conducted by the present inventors has shown that 'I' N F or healthy individuals are found in the body fluids of patients suffering from Kawasaki disease and bacterial infections, for which it is extremely difficult to diagnose the disease and check its progression, as will be described later. It is contained in larger amounts than humans, and its 'I' N
It has been found that the amount of I' and its changes are closely related to the progression of these pathological conditions. Therefore, it is extremely important to accurately and quickly measure the content of 'rN17 in the body fluids of patients with these diseases in order to check the progress of their pathological conditions.

Kawasaki disease (acute febrile mucocutaneous lymph node syndrome; Infan
tile acute fcbrile ll1uco
cutaneous-1y+nphnode-synd
roIIle; MCI, S) was published in Kawasaki in 1967 ['l', Kawasaki, “7 Lurugi, ■, 17
8 (196711) It is a disease of unknown cause that frequently occurs in children, and includes fever that lasts for more than 5 years, hard edema of the hands and feet, erythema on the heel or tip of the finger, membranous desquamation from the fingertip during the recovery period, and l- 1 Dry lips, red face, cracks, strawberry tongue 1 Redness of oral mucosa.

Rash on body ↑, hyperemia of bulbar conjunctiva, cervical lymph node II! i
It is a disease characterized by many symptoms such as dizziness.

The diagnosis of Kawasaki disease is based on ['I', Kawasaki, "Pediatrics" 26°985 f1985)], but the etiology is unknown and the disease is diagnosed based on a combination of major symptoms. It is an extremely difficult disease to make a definitive diagnosis. In particular, the formation of coronary artery aneurysms in this disease is the main cause of death in this disease, and it is urgent and important to find indicators of pathological conditions that can predict the formation of coronary artery aneurysms in advance as Kawasaki disease progresses. Na;i′! It was a question.

The present inventors measured body fluids (e.g., serum) in patients with Kawasaki disease, and found that 'r' in the serum of patients with Kawasaki disease with severe symptoms that may lead to coronary artery aneurysm formation.
It was found that NF was significantly detected.

Furthermore, the present inventors conducted JIIJ measurements on body fluids (e.g., serum) of patients with bacterial infections, and found that the number of white blood cells in them,
Corresponds well with changes in the amount of platelets and C-reactive protein, and T
It has been found that the detection of NF and, therefore, the measurement of r'NF' in body fluids in the diagnosis of the symptoms of bacterial infections is extremely meaningful in the diagnosis and in determining the progression of the disease state.

The present invention was achieved based on the above-mentioned new findings, and is based on the immunological 4 (11
By detecting an anti-tumor necrosis factor antibody-reactive substance in a body fluid of a subject, characterized by detecting an anti-tumor necrosis factor antibody-reactive substance in a body fluid collected from a subject by a standard method. This is a detection method for determining the pathological condition of a subject.

Thus, according to the present invention, body fluids of a diseased patient or a subject suspected of having the disease are collected, and an antitumor 't1!21 death factor antibody-reactive substance disposed therein is collected. By measuring the content of tumor necrosis factor ('r'N)'' and detecting the content or change thereof, it is possible to judge the progression of the disease state.

In particular, the present invention is advantageously used for determining the pathological condition of patients with Kawasaki disease and bacterial infections, as described above.

In the present invention, body fluids from a subject are collected and anti-tumor necrosis factor-reactive substances therein are detected by immunoassay using anti-11i1'ty islet necrosis factor antibodies. ,
The body fluid may be any liquid component present in the human body that can be collected, and blood components such as serum or plasma are particularly desirable.

However, in addition to blood components, exudate, lymph fluid, synovial fluid, etc. at the inflamed site may be used, or blood components in and near the affected area may be used, and T' N F-' can be determined by frequent measurements.
When examining changes in ffi, it is desirable to use blood components of peripheral blood obtained by a normal collection method, but the method is not particularly limited thereto.

In accordance with the present invention, the resistance n! To measure tumor necrosis factor-reactive substances by immunoassay,
An anti-tumor necrosis factor antibody is used, and the antibody is an antiserum or a monoclonal antibody against tumor necrosis factor ('T'Nl'').

As such an immunoassay method, the sandwich method, which is generally known per se, is advantageously used. In immunoassays using the sandwich method, two types of antibodies are usually used, but even when implementing the detection method of the present invention, one of the two types of antibodies is an anti-tumor necrosis factor monoclonal antibody, In particular, it is preferable to use an anti-human tumor necrosis factor monoclonal antibody because 'T' NF can be detected accurately and quickly.

The sandwich method that is advantageously used as the immunoassay method of the present invention is a known method that is often employed to detect a specific protein of V&K using an antibody. Described in the literature.

langone, J., and van Vunak.
is, 11. , (ads,) :Methods
in [nzymology, Vol, 73. Iln
+unochemical techniques Pa
rtB, ^ academic Press, New Yo
rk.

Nakamura, Il., H., Dito, W., R. an.
d luckar, E, S,, 11 (OdS,): 1
vaunoassays in the Cl1nic
al Laboratory, Alan R.
, New York, 1979Ish+kav+a,
E, , niawai, T, and Hiyai, ni,
(cds, ) :Enzyme 1m1unoass
ay Igaku-3hoin, Tokyo, 1981
As mentioned above, the present invention aims to reduce the amount of anti-I contained in the body fluid of a subject.
i! The disease condition of the subject is determined by detecting the 1ir7d necrosis factor-reactive substance by immunoassay, and the disease state or suspected disease includes antitumor fsJ11 death factor-reactive substance, Specifically, T N
6 Kawasaki disease or bacterial infection is a typical disease, and the amount of F in the body fluid of the subject is higher than that observed in a healthy person as a result of the disease. TN
F? The content and its changes are important criteria for determining the pathological condition.

However, even if it is a disease other than Kawasaki disease or a bacterial infection, if the content of 'r'NF is related to the judgment of the pathology of the disease, the detection method of the present invention can be used to detect the disease. is used in the same way.

The above-mentioned bacterial infection refers to a disease in which bacteria invades and multiplies within a living body, causing the living body to exhibit a pathological condition, and is a general term for an extremely diverse range of diseases and pathological conditions6.
For example, they are classified as systemic infections such as sepsis, local infections such as urinary tract infections, respiratory tract infections, otorhinolaryngological infections, and gastrointestinal infections, but in reality they are caused by a variety of different pathogens. It is difficult to classify the disease because it often exhibits symptoms of one disease. Symptoms of these bacterial infections are extremely diverse and often lead to serious conditions. Symptoms such as erythema may also be observed, and symptoms such as hypotension, shock symptoms, embolic symptoms, and bleeding tendency may also be observed, but in many cases there are no specific symptoms, making it difficult to diagnose the severity of the condition. There was too.

The present invention is useful for monitoring the pathological condition of these bacterial infections as long as it is meaningful to measure the content of '1'Nl) in body fluids depending on the pathological condition.

Therefore, the present invention is applicable not only to Kawasaki disease and bacterial infections, but also to pond diseases as long as the TNF site in body fluids shows an abnormality due to changes in the @ state as a result of the disease; other diseases include, for example. systemic erythromatosus, autoimmune diseases such as rheumatoid arthritis, sarcoidosis, ulcerative colitis, chronic inflammatory diseases such as Crohn's disease, congenital and acquired immunodeficiency disorders,
Immune dysfunction associated with transplantation, such as rsus bost disease), and other widespread vasotropic coagulation (desse
n+1uated 1ntravascular co
Examples include diseases accompanied by vasculitis such as vasculitis.

According to the present invention, there is provided a method for accurately and rapidly detecting an anti-tumor necrosis factor-reactive substance in a subject's body fluid by an immunoassay method using an anti-tumor necrosis factor antibody, and the method therefor. kit is provided.

Subject's T N l? Accurately detecting the content is
It is necessary for accurate judgment of the disease state, and if detection is delayed, there is a risk that as the disease progresses, we will lose time to use the detection results to prevent the progression of the disease or treat it.

Therefore, the anti-tumor necrosis factor antibody of the present invention includes anti-I11
′! A 5% necrosis factor monoclonal antibody is used for the above purpose.

One type of anti-tumor necrosis factor monoclonal antibody is described in Japanese Patent Publication No. 1988-208924 and is publicly known, and the monoclonal antibody described in this publication is human
Although it is characterized by binding to human 'I' NF and inhibiting its activity, the publication does not provide any equivalent explanation as to which site of human 'I' NF it binds to.

According to the research of the present inventors, the detection method of the present invention includes the following (
Using anti-human tumor necrosis factor monoclonal antibodies characterized by a1-(c), '
It has been found that I' N F can be detected accurately and quickly.

(a) Has the ability to neutralize the killing effect and fatty acid metabolism inhibitory effect of human tumor necrosis factor on L929 cells, (b) The 68th amino acid sequence of human tumor necrosis factor
GIV) to 97 IHIIe), and (c) specifically inhibits the binding of human tumor necrosis factor to the tumor necrosis factor receptor.

As far as the present inventors know, the anti-human tumor necrosis factor monoclonal antibodies characterized by (a) to (c) are novel and provided for the first time in the present invention (hereinafter referred to as "this monoclonal antibody"). ” MCA-
(sometimes abbreviated as "A").

Further, according to the present invention, human tumor necrosis factor (human 'I'
Anti-human 'f' that recognizes the site included in the amino acid sequence 7th (Thr) to 37th (Leu) of NF )
Also provided is a NF monoclonal antibody (hereinafter this antibody may be abbreviated as "MCA-B"),
This MCA-B did not neutralize the cell killing effect of human TNF on L929AIII cells.

The fatty acid metabolism inhibitory effect of human i'NF also has the property of not being neutralized.

Furthermore, according to the present invention, amino acid sequence position 113 (Pr
o) to 127 t:I (Glu) are provided (
(Hereinafter, this antibody may be abbreviated as 'MCA-C').

This MCA-C has the property of not neutralizing the cell-killing effect of human 'I''NF on L929 cells, nor the fatty acid metabolism suppressing effect of human 'r'NF, and
It does not inhibit the binding of human 'I' NF to NF receptors.

The above-mentioned MCA-A, MCA-B and MCA-C are
All of them are novel monoclonal antibodies against human i' NF, and can be used as at least one type of anti-i' NP antibody in the detection method and kit for detection of the present invention, but MCA-A is particularly useful. , is the most desirable from the viewpoint of connectivity.

Below, the kit for preparing and detecting the monoclonal antibody against human 'f' NF of the present invention will be explained.

(Preparation of anti-'I' NF antibody) (1) Preparation of anti-'I' NF antibody-producing cells Antibody-producing cells may be prepared according to a conventional method. That is, the antigen, human)-' A method may be used in which an animal is immunized with 1'N I'' and antibody-producing cells from the animal are obtained.

Animals include mice, rats, rabbits, guinea pigs,
Examples include sheep, horses, and cows, and III! ! Cells, lymph node cells, peripheral blood cells, etc. are used.

(2) Bone marrow used in the cell fusion method for preparing myeloma cells +1. Ij cells are not particularly limited, and many animal cell lines such as mice, rats, rabbits, and humans can be used.

The cell line used should preferably be drug resistant so that unfused bone marrow lung cells cannot survive in the selective medium and only hybrid cells will proliferate. The most commonly used cell line is 8-azaguanine resistant, which is deficient in hyboxanthin guanine phosphoribosyl transferase and cannot grow in hyboxanthin-aminopterin-thymisin (HA T ) medium. has. In addition, it is preferable that the cell line used is a so-called "non-secreting" cell line, for example, the mouse myeloma line MOPC=21 Yamaki's P3/X63 A.
g8LJ1 (P3U1)P:l/X63A
g・6・5・3, P, /N51-1-Ag4-1,
Sρ210-Ag14, rat myeloma cells 210RCY
3.Agl.2.3 etc. can be suitably used.

(3) Single-cell fusion normal Eagle minimal essential medium (MEM), Rosewell Park Memorial Institute (R,RM
I) 1-5 x 1 in a medium such as 1640 medium
Bone marrow l1lI between 07! cells and antibody producing cells 1-5 x
1 (mixing 1811U (mixing ratio is usually 1:4 to 1:1)
0), cell fusion is performed. As the fusion promoter, polyethylene glycol (PEG) having an average molecular weight of 1,000 to 6,000 is preferred, but viruses and the like can also be used. The concentration of PEG used is usually 30-50%.

(4) Selective proliferation of hybrid cells A
Seed in microtiter plates at approximately 105 ml.

Add selective medium (e.g. HAT medium) to each well;
Thereafter, the selection medium is appropriately replaced and cultured. Myeloma 4:■ If an 8-azaguanine-resistant strain is used as one cell, all unfused myeloma cells will die within about 10 days in HA'r medium, and antibody-producing cells are normal cells, so in vitro They cannot grow for a long time (in vitro), therefore, all cells that grow after about 10 to 14 days of culture are hybrid cells.

(5) Search for antibody-producing hybrid cells Screening for hybrid cells may be carried out by conventional methods and is not particularly limited. Human 'I'
After reacting with NF, the enzyme, radioisotope,
By reacting with a second antibody labeled with a fluorescent or luminescent substance, the amount of labeling can be determined and the presence of anti-human 'r'N I;' antibodies can be assayed.

(6) Cloning Two or more types of hybrid cells are growing in each well II
Therefore, cloning is performed using limiting dilution method to obtain 1 monoclonal antibody-producing hybrid cell.6 (7) Obtaining the antibody The purest monoclonal antibody can be obtained by cloning the desired hybrid cell by
It can be obtained from the culture supernatant obtained by culturing in an appropriate culture medium such as RPM or 11640 medium containing about 10% fetal bovine serum.

On the other hand, in order to obtain even larger amounts of antibodies, pristane (2, 6, 10
, 14-tetramethylpentadecane) by intraperitoneal administration of mineral oil, and then administering the f & hybrid cells.
Hybrid cells can be grown in large quantities in vivo.

In this case, an ascites tumor is formed in about 10 to 18 days, and high concentrations of antibodies are produced in the serum and ascites.

(Kit for detecting human TNF and preparation thereof) The monoclonal antibody against 'I' NF obtained as described above is used as at least a part of the anti-i' NF antibody in the detection method and detection kit of the present invention. used.

In immunoassays, particularly in the sandwich method, two types of antibodies are generally used that have binding properties to one antigen (e.g. protein); Species antibodies are used, at least one of which is a monoclonal antibody against the human 1"N I". Are the two antibodies both human 'rNI?
One type of antibody may be a monoclonal antibody that recognizes different sites against human ``[''NF'', and the other antibody may be an antiserum against human TNF''.

Next, to explain a kit using two types of antibodies against human 'I' NF, an antibody against human '1' NF (first antibody) is immobilized on a suitable insoluble solid carrier (for example, a plastic container). (hereinafter referred to as "immobilized antibody"), and then an appropriate substance (for example, bovine serum albumin) to avoid non-specific T4 binding between the insoluble solid carrier and the serum or other body fluid sample to be measured, that is, the specimen sample. The surface of the insoluble solid support is coated with

The insoluble solid carrier on which the first antibody thus obtained is immobilized is brought into contact with the specimen sample for a certain period of time and at a certain temperature to cause a reaction. During this time, the immobilized antibody (first antibody) and 'T' N F in the specimen sample are bound together. 6 Then, after washing with an appropriate washing solution, human 'I' N F labeled with an appropriate labeling substance is bound to the immobilized antibody (first antibody).
A solution (e.g., an aqueous solution) of an antibody (second antibody) to an insoluble solid 'I' bound to an immobilized antibody in one body.
It is brought into contact with NF for a certain period of time and at a certain temperature to react with the second antibody. This is washed with an appropriate washing solution, and then the residue of the labeling substance labeled with the second antibody present on the insoluble solid support is determined. Thus, from its value, i' in the specimen sample
The detection kit of the present invention, which is capable of calculating the amount of NF, is mainly composed of a first antibody or an immobilized antibody bound to an insoluble solid support, and a labeled second antibody. In order to utilize this kit efficiently and easily, it is possible to form a kit containing various auxiliary agents in addition to these antibodies. Such auxiliary agents include, for example, solvation agents for dissolving solid reagents, detergents used for washing insolubilized solid supports, and when enzymes are used as labeling substances for antibodies. Examples include those commonly used as kits for immunoassay reagents, such as substrates for measuring enzyme activity and reaction terminators.

Examples of the insoluble solid carrier used in the detection kit of the present invention include borisdylene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin,
Examples include polymers such as cross-linked dextran polysaccharide, paper, glass, metal, agarose, and combinations thereof.

The shape of the insoluble solid carrier is, for example, a tray, a sphere, a fiber, a rod, a disk, or a container.

It can be in the shape of a persimmon, such as a cell or a test tube.

It is also advantageous to use radioactive substances, enzymes or fluorescent substances as labeling substances. Radioactive substances include I,
I, C, H, etc., alkaline phosphatase, peroxytase, β-1)-calactosidase, etc. can be used as enzymes, and fluorescein inthiocyanate, tetramethylrotamine isothiocyanate, etc. can be used as fluorescent substances. However, these are not limited to those exemplified, and other types can be used as long as they can be used in immunological measurement methods. Furthermore, an auxiliary agent can also be used to increase the sensitivity of the labeling substance.

In the detection method and kit of the present invention, the anti-
The r' NF antibody is used as the first and second antibody.

That is, among the plurality of monoclonal antibodies prepared by the above method, two that give good results as a quantitative measurement system are selected.
Different types of monoclonal antibodies are selected and used as the first and second antibodies. Alternatively, anti-human 'I' NF antiserum can be obtained by a conventional method using a warm-blooded animal and used as either the first antibody or the second antibody.

EXAMPLES Hereinafter, the present invention will be explained in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 (Preparation of anti-human i' NF monoclonal antibody) Higeomu Kasasui The method for producing human 'I' NF used in the present invention is described in a previously issued patent (Japanese Patent Application No. -No.90087:
The method described in April 21, 1986 [1. Jaw protrusion 2. Name of the invention "Novel physiologically active polypeptide'"] was used.

That is, E. coli into which the human 'I' NF gene expression vector was introduced was cultured, and P1 to TN1? Promoted protein production. After collection, E. coli was disrupted using ultrasound, and the resulting suspension was used by 5hirai et al. [T, 5hirai et al.
Va was produced by DEAE seoharase column chromatography according to the method of Nature, 313.830 (1985)]. 'I' in this crude product
The NF content was approximately 30%.

50 to 100 tt g of the above 'I' NF fraction made into an emulsion in 100% complete adjuvant to human and 'r゛NF fractions was subcutaneously administered to Ba1b/c mice for 2 weeks. Four days after the final immunization, the II'!' viscera was removed and used for A11I cyst lysis.

Cell fusion 4: The amount of bubbles per mouth was determined according to a conventional method. That is, a cell suspension was prepared from the aseptically removed spleen through a mesh, washed three times with RPM11640 medium, and then P3-X63-Ag8-Ul cells (abbreviated as P3U1), which are mouse myeloma cells, were prepared. ) [D, E, Yalt
onCurrent Topics in Hicr
obiology and I+onunolay, 8
1.1 (1978)] in a ratio of about 1 to 5:1, and after centrifugation, add 50% polyethylene glycol 1540. RP MI-1640 thawing 1m
1 was gradually added and the centrifuge tube was slowly stirred for 1 minute to perform cell fusion. Furthermore, Ft PMI-164
0 medium gradually over time until finally 10ml
And so. After centrifugation, the pellet was placed in RF containing 10% fetal bovine serum.
'5 to 10 myeloma cells in MI 164G medium
The suspension was suspended in a volume of 104 x 104 cells (10.1 ml), and 0.1 ml each was inserted into a 96-well Mic 17 plate (Costar).

One day later, 0.1ml of HA'i'' medium was added to each well, and after that, half the volume was replaced with HA'r'' medium at appropriate intervals. Growth of hybrid cells was observed in the wells, and after 21 days, hybrid cells proliferated in almost all the wells.

Take 0.1 ml of the culture supernatant from the well where the antibody-producing and cloning hybrid cells have been grown, and ink and combine with human 'f'Nr' immobilized on an immunoassay plate (Titertic). When we looked for something that could bind to human 'I' N I', we found wells that secreted antibodies capable of binding to human 'I' N I' with a probability of about 40%.

Only a portion of cells with high binding ability were selected, and cloning was performed by the limiting dilution method in which cells were planted in a 96-well microplate at a density of 1 cell/well.

Some of the obtained clones, 93, were selected and 10
Using RP MI 1640 medium containing % fetal bovine serum, the culture was scaled up sequentially from 96-well plate to 24-well plate to 6-well plate 1 to 25-flask, and the culture supernatant was collected.

Each supernatant was mixed with 1000 units/m+ human'['Nl
? After mixing the warm solution and incubating at 37°C for 1 hour, I.

T' N F' activity was evaluated using 929 cells, and 11 clones (9C4G5.8E6B6, 10871', it, 1
1D7G4゜8E6C6,2B2) [to, 9C
4A9. 8E6D7, IG7D3, 81 Shiro G4,
1087C6), it has little ability to neutralize
'5 clones with high binding ability to NF (9E8G7.l
A10D4゜IF12A7. IF12A9. P4B4D
5) Selected.

Monoclonal r° talent I! Next, the culture of the cells of the 16 clones was further scaled up using R''P M 11640 medium containing 10% fetal bovine serum, and approximately 500ml to 10ml of culture supernatant was collected.This culture supernatant was brought to 50% saturation. After stirring with ammonium sulfate at 4°C for about 1 hour, centrifugation was performed at 10,000 x G for 30 minutes.The pellet was dissolved in pure water and dialyzed against 0.1M phosphate buffer D) 18.0. This solution was applied to a column of Protein A Sepharose (Pharmacia), and the adsorbed monoclonal antibody was eluted with 0.1M citrate buffer of 1115.0 or pi (3.0), neutralized with NaOH, and then passed through a membrane filter. (Amicon YM-10) and substituted with 0.1M phosphate buffer ol-18.0 to obtain a purified monoclonal antibody solution.

Example 2 (Confirmation of TNF-specific recognition ability) The above crudely purified i'
NF and purified 'r'N I'' to 5DS-PAG
After electrophoresis was performed at P[, AT E 10/20 (First Chemical FM), plotting buffer (
The cells were blotted onto nitrocellulose filters in 20mM Tris, 150nM glycine, 20% methanol). Nido 17 cellulose filter was filtered using a buffer (201M Tris) containing 3% gelatin.

After shaking for 1-2 hours at room temperature in 500+ nM NaCf) purified antibody was purified at 2 μft/[+11 and 1%
The buffer was changed to a gelatin-containing buffer and shaken overnight at room temperature. Washing solution (201M Tris, 500mM Na
After washing the Nitrocell one-course filter three times with CF, 0.05% Twcen 20), goat anti-mouse +
aa, HRP complex itself (1310-RAD company),
It was diluted with a buffer containing 1% gelatin to the dilution ratio specified by the manufacturer, and the Nido ■7 cellulose filter was shaken therein at room temperature for 1 to 2 hours. 3 with the cleaning solution
times.

and 'I'ween20-free washing solution, IIRP DevelopIIlent Rea
Aent (BIO-RAD) was used to develop color. An example using IF12A7 as a purified antibody is shown in FIG.
As is clear from this figure, in both crude 'I' N F' and purified TNF, IF"12A7 binds and i' N
I? It was confirmed that it has specific binding ability. Similarly 9C4G5゜8E6136, 10E17E11
,11D7G4.8E6C6,2B2H10,9C4A
9,8E6D7. IG7D3,8E6G4.10r17
C6,9E8G7゜lA10D4. IF12A9. P4
It was confirmed by this method that each B4D5 antibody had specific binding ability to i'NI'.

Example 3 (Study of ability to neutralize fatty acid metabolism inhibitory effect) Mouse fibroblasts 3′r3-L] were incubated with C0nflLI+311
115 μg/rnl of MIX after growth at
(3-isobutyl-+actyl-xant
hina), 395 ng/ml I)
clexaIIlethasone), 10μt/
L M D M E11f containing 10% fetal bovine serum containing 1 ml of INS (bovineinsulin)
At the i site, differentiation induction was performed for 2 days to differentiate into adipocytes. Two days after induction, the differentiation medium (containing 10% fetal bovine serum containing 50 nQ/ml INS, MDMEM) was added.
After 1 to 2 hours, the sample was added.

As a sample, TNI? plus 'rNF and anti'
T'NF antibodies were mixed at a molar ratio of 1:1, incubated at room temperature for 1 hour, and then 'I' N
47n (1/ml) was used as the FJ degree. After adding the sample, it was incubated at 37°C for 4 hours, then heparin was added to a final concentration of 10 U/mount, and the mixture was incubated for another 1 hour. After incubation, the supernatant was collected and the 1,1poprotein Lipase activity contained in the supernatant was
It was measured by a reaction using triolein labeled with F■ as a substrate. As shown in Figure 1 below, the results showed that anti-i' that can neutralize the cell-killing effect of 'l' N F against [929]
The NF antibody is 3'r31. due to 'f' N r'.
It has been revealed that it also neutralizes the inhibitory effect on fatty acid metabolism in adipocytes.

In addition, the anti-TNF antibody that does not neutralize the cell-killing effect of 'I' N I' on L929 is
'3-1.1 It was revealed that the inhibitory effect on fatty acid metabolism in adipocytes was not neutralized.

Table 1 Example 4 (Identification of specific recognition site) Based on the known TNF amino acid sequence, a fragment peptide having the i' NF amino acid sequence shown in Table 2 was synthesized using a peptide synthesizer from Applied Bio Systems. Synthesized using 43OA.

The peptides dissociated from the supporting resin by hydrogen fluoride treatment after 1 h are subjected to purity assay and, if necessary, purification using reversed-phase column chroma 1 to Graphie, and are purified to a sufficient mV! ! The one for which the degree was obtained was used in the experiment.

Purified peptide fragments (0.5-1 μ/ml) were
It was left to stand overnight at 4°C on a JI immunoassay plate (Titertic)-L for immobilization. 0.5% raw blood? I'
Cleaning solution containing albumin (20+1 phosphate buffer)
-0,135M Nacf, 0.05%'l'
After washing three times with wean 2002% NaN5),
Buffer containing % bovine serum albumin (20+aM phosphate buffer T-, 0,135M NaCl, 0,2% N
aN3) was added and left at room temperature for 1-2 hours. After washing three times with the washing solution, purified anti-TNF antibody was added and reacted at room temperature for 1 to 2 hours. After further washing three times with the washing solution described above, goat anti-mouse 1 (IG-alkaline phosphatase complex) was added, and further 1 to 2
I left it for a while. After washing with the washing solution, alkaline phosphatase substrate, p-N1trophenyl Ph
phate and Disodiutn were added at a concentration of 1/ml, and the change in absorbance per minute at a wavelength of 405r++n was measured using TLISA ANALYZER (Toyo Sokki ■1': 'rY'-96).Reactions to 6 different peptides. When the binding properties of each antibody were examined using the method described above, each antibody exhibited binding properties as shown in Table 3.

It was found that the antibody that neutralizes the binding 'r' NF activity exhibits specific binding to a peptide having the amino acid sequence from position 68 to position 97 and 11 of '1' NF shown below.

Nl+2 -GIV Cys Pro Scr
rhr Vat tau leu Thrs
rhr lie Ser Arg IIe ^
la Val Ser ryr Glnrl+r
lys Val Asn tau leu Ser^l
Three clones (IF12A7.IF1
2A9,9E8G7) antibody binds to D in Table 2 shown below and ■, which is the 'r' in the common part of Amane.
N I? It was found that the peptide showed specific binding to the peptide having the amino acid sequence from the 113th to the 127th amino acid sequence.

Table 2 Self-adjudication peptide Nt12Pro Trll ryr Glu Pro
Ile lyr LeuGly Gly Vat
Phe Gln tau Glu -COO
IA10D4, which is incapable of neutralizing the activity of II or i'NF, is not listed below.
Pepti 1, which has the amino acid sequence from No. 7 and No. 11 to No. 37 of Nl'', showed specific binding.

Ntl□−’lt+r　Pr.

Val Va G n 1. eu 8sn A a Sar 8sp It, IS Pro Vat
^1atlisAla Asn Pro Gln
^la Glu GlyGln Trp le
u Asn Arg Arg Alal, eu
1. Binding of untested partial antibody to synthetic peptide for eu-COOIIN3 Next, the following experiment was conducted to clarify the site where the antibody binds to 'I' NF.

Purified 1'NF (1 μg/ml) was placed in an immunoassay plate (Titertic) at 4°C overnight.
Fixed. Cleaning solution containing 0.5% bovine serum albumin (2
0+++M phosphate buffer +, 0.135M
Na, CI.

0.05%'I"weenlo, 0.2%Na
After washing three times with N3), buffer γ-(20+nM phosphate buffer y+, (
1,135M N ac,i'.

Add 0.2% NaN:+) and incubate at room temperature for 1 to 2 hours.
I left it alone. Said washing? After washing with sound three times,
The anti-1' NF antibody and the hexagonal peptide were prepared in advance.
A mixture that had been pre-incubated for 1 hour at 7°C was added and allowed to react for 1-2 hours at room temperature. Furthermore, the above
After washing with washing solution 3 (η1), goat anti-mouse xga
-Alkaline phosphatase complex was added and the mixture was further left at room temperature for 1 to 2 hours. After washing with the washing solution, the alkaline phosphtase substrate p-N1tropt+eny
l Phphate, mouth 1 sodi + n++ 1 m
g/ml concentration, ELISA ANAI,
Y Z E R (E i' Y-9 made by Toyo ap I u
In step 6), the change in absorbance per minute at a wavelength of 405 nm was measured.

No. 21: A is monoclonal antibody I F 12A 7
For binding with TN-do, hexagonal peptide D (see Table 2)
This study showed that the drug was inhibited in a dose-dependent manner.

The '1' of the antibody when the peptide is added is set as 100% when the antibody's 'l' NF binding without the synthetic peptide is added.
゛Binding with NF is shown as a percentage. From this result, monoclonal antibody IF12A7 is 1-ys to 98th.
The part contained in Glu・27 is used as the binding site 'I
It was found that it is combined with 'NF'.

Example 5 (Impact of antibody on binding of 1'NF to "I' N I" receptor g) [nzy+oobcacls (BIO-ltAI1 company)
The specific radioactive activity of TNFC introduced with 1 is 1.2 x
1010 CPm/gprotein) to the i'NF receptor on L929 cells.
The effect of anti-'f' NF antibody on l-'f' NF binding was investigated. 5 x 10' L929 AI cells
Seed on dates to form cells and incubate at 37°C.
After incubation for 4 hours, discard the supernatant.
51-'I'NF (51n (1/ml) or I-'f'NF (51nlJ/ml)
and anti-'f' NF antibody (5,1u f/onl)
The sample was incubated at room temperature for 1 hour and then added to the cells. On the other hand, to ensure specific binding to the receptor, each sample was
Non-Ii of Baiji? The i-identifier 'f' NF was added to 1,929 cells in the same manner. After incubation at 4°C for 5 hours, the cells were collected with a scraper, washed twice with a medium (Eagle's medium containing 5% tallow serum) by centrifugation, and then separated into 4.1 foam.
The count was measured. As an example of the result, 'I' N
Anti-TN1・' antibody that neutralizes the activity of F.111)71
The results when using the above are shown in Table 4 below. From this result, T
Antibody 11D 7 G 4 of NFl' is 'I' NF i' Nl? It is clear that binding to the receptor is not inhibited at all.

In addition, 4 clones (IF12A7, 1.F12A9.9E8G7.
1A10D4), and the active neutralizing antibody 1087 F, which inhibits the binding of 'f' NF to the 'I' NF receptor.
The results when using No. 11 are also shown in Table 4 below. From these results, the anti-T NF antibody IF12A7, which recognizes the region from the 113th to the 127th amino acid sequence of 'I' NF, is TN It is clear that there is no inhibition of binding to the F glue receptor.

Table 4 Example 6 (Creation of standard curve) The antibody used in this example was “11D 7 G” among the monoclonal antibodies produced by the clone described in Example 1.
9C4G5'° was labeled with bovine liver-derived alkaline phasphatase (Sigma) and used as a secondary antibody.

Monoclonal antibody (IID) at a concentration of 15 μg/onl
7G4) was immobilized on an immunoassay plate (TiterDeck) by leaving it at 4°C overnight. Wash Kf containing 0.5% live serum albumin (20n+M phosphate buffer 0.135 M Nacf, 0.05
%'f'ween20 0.29≦N2LN:]) and 3
After washing twice, buffer T-(201 nM phosphate buffer 0.135
M NaCl, 0.2% Na, N3) was added and left at room temperature for 1 hour. After washing three times with the washing solution, various concentrations of human i''NF were added and reacted at room temperature for 1 hour. After washing three times with the washing solution, a secondary antibody labeled with alkaline phasphatase (9C4
G5) was added and reacted at room temperature for 1 hour. After washing with the washing solution, alkaline phosphaturbi substrate p-N1t
rophenyl PhosDhate, formerly sodi
ELISA by adding um at a concentration of 1 mg/m+
ANALYZER (Toyo nj equipment @ J made E i' Y -
96), the change in absorbance per minute at a wavelength of 405 nln was determined as JjlJ. The results are shown in Figure 3 of the attached drawing. From this figure, the amount of human TNF can be easily measured by the Sand-Deutsche enzyme antibody immunoassay method using a monoclonal antibody that specifically recognizes human 'rNF.

Example 7 (Measurement of T'N I7 in Kawasaki disease patient serum)
Sera from Kawasaki disease patients and serum from healthy people and patients with other diseases as controls were added to a phosphate buffer (20+nM phosphate buffer).
-, 0,135M NaC11187,2), 2~
It was diluted 10 times, and the amount of 'f' NF was measured using the sandwich method described in Example 6 using the antigen. A calibration curve was created at the same time and under the same conditions.

The results are summarized in Figure 4 of the attached drawings. T'NF was detected in serum in 12 of 30 Kawasaki disease cases (40%). There was a significant correlation between the detection of 'I' NF and the appearance of coronary artery lesions, as shown in Figure 5 of the attached drawings.

In addition, as shown in Figure 6 of the attached drawings, 'T'NF is a disease commonly observed in patients with severe symptoms during the acute phase.
Measuring the amount of 'I'NF using the measurement kit according to the present invention is extremely effective for diagnosing Kawasaki disease and analyzing its pathological condition, especially for pre-diagnosing the appearance of coronary artery lesions.

'l' corresponds very well with the severity of the infectious disease.
It can be seen that there is a high correlation with N F ffi. Therefore, measuring the amount of 'I'NF using the measurement kit according to the present invention is highly effective in the diagnosis of M1 bacterial infection and the analysis of pathological conditions.

Table 5 Detailed Table Measurement of 'r'NF' Amount in Serum of Person A Example 8 (Measurement of TN F in Serum of Patient with Bacterial Infection) Serum of patient with Phlebrasiella numeremia as bacterial infection and
As a control, serum from healthy people and patients with other diseases was mixed with phosphate buffer (20mM phosphate buffer, 0.135M Na
'I' N
I"Ji was measured, and a calibration curve was prepared at the same time under the same conditions. The results are summarized in Table 5 below.

From Table 5 below, TNI'ffi in a patient's serum is associated with changes in the number of white blood cells, number of platelets, and amount of C-reactive protein in the same blood.

[Brief explanation of the drawing]

Figure 1 shows 'I' of anti-'f'NP monoclonal antibody.
This shows the binding property of NF. FIG. 2 shows the inhibitory effect of synthetic peptide D (see Table 2) on the binding of monoclonal antibody IF12A7 to i'NF'. FIG. 3 shows a calibration curve for human i' NF measurement according to the present invention. FIG. 4 shows the results of measuring the amount of TNF in the serum of Kawasaki disease patients and control rats according to the present invention. Figure 5 shows the relationship between the serum hydration level of 'I' N F and the appearance of coronary artery lesions, and Figure 6 shows the relationship between the fever type pattern, white blood bead count (WBC), and platelet count (
The figure shows changes in the contents of P L 'r) and 'r' N I'' and the appearance of coronary artery lesions over time.

Claims (1)

[Claims] 1. By immunoassay using anti-tumor necrosis factor antibody,
Detecting an anti-tumor necrosis factor antibody-reactive substance in a body fluid collected from a subject. Pathological condition of a subject by detecting an anti-tumor necrosis factor antibody-reactive substance in a body fluid of a subject. Detection method for determining. 2. The method according to claim 1, wherein the anti-tumor necrosis factor antibody is an anti-human tumor necrosis factor monoclonal antibody. 3. The anti-human tumor necrosis factor monoclonal antibody (a) has the ability to neutralize the cell-killing effect of human tumor necrosis factor on L929 cells and the fatty acid metabolism inhibitory effect, and (b) has the ability to neutralize the amino acid sequence of human tumor necrosis factor. It recognizes the site contained in positions 68 to 97, and (c) specifically inhibits the binding of human tumor necrosis factor to the tumor necrosis factor receptor. Claim 2, which is an anti-human tumor necrosis factor monoclonal antibody.
Method described. 4. The method according to claim 1, wherein the subject is a Kawasaki disease patient. 5. The method according to claim 1, wherein the subject is a patient with a bacterial infection. 6. The method according to claim 1, wherein the immunoassay is a sandwich method. 7. The method according to claim 1, wherein the body fluid is serum or plasma collected from the subject. 8. An anti-human TNF monoclonal antibody capable of neutralizing the cytocidal effect of human tumor necrosis factor (human TNF) on L929 cells. 9. The monoclonal antibody according to claim 8, which is capable of neutralizing the fatty acid metabolism inhibitory effect of human TNF. 10. The monoclonal antibody according to claim 8, which recognizes a site included in amino acid positions 68 to 97 of the human TNF. 11. The monoclonal antibody according to claim 8, which specifically inhibits the binding of human TNF to a TNF receptor. 12. Anti-human T that recognizes the site matching amino acid positions 7 to 37 of human tumor necrosis factor (human TNF)
NF monoclonal antibody. 13. The monoclonal antibody according to claim 8, which does not neutralize the cell-killing effect of human TNFC on L929 cells. 14. The monoclonal antibody according to claim 8, which does not neutralize the fatty acid metabolism inhibitory effect of human TNF. 15. An anti-human TNF monoclonal antibody that recognizes a site included in amino acid positions 113 to 127 of human tumor necrosis factor (human TNF). 16. The monoclonal antibody according to claim 15, which does not neutralize the cell-killing effect of human TNF on L929 cells. 17. The monoclonal antibody according to claim 15, which does not neutralize the fat fatty acid metabolism inhibitory effect of human TNF. 18. The monoclonal antibody according to claim 15, which does not inhibit the binding of human TNF to the TNF receptor. 19. It consists of a first anti-tumor necrosis factor antibody, a second anti-tumor necrosis factor antibody, and an insoluble solid carrier, and one of the antibodies is immobilized on the solid carrier. A detection kit for detecting an anti-tumor necrosis factor antibody-reactive substance in a subject's body fluid and determining the disease state of the subject. 20. The detection kit according to claim 19, wherein at least one of the first anti-tumor necrosis factor antibody and the second anti-tumor necrosis factor antibody is an anti-human TNF monoclonal antibody. 21. At least one of the first anti-tumor necrosis factor antibody and the second anti-tumor necrosis factor antibody is capable of neutralizing the cytocidal effect of human tumor necrosis factor (human TNF) on L9294 cells. Claim 1: The antibody is a monoclonal antibody.
9. Detection kit according to 9. 22. The detection kit according to claim 19 for determining the pathology of Kawasaki disease.